The molecular pathogenesis of fully virulent, wild-type Y. pestis in relevant animal models has been relatively neglected because of the scarcity of secure BSL-3 facilities and trained personnel certified to work with this Class A select agent. The threat of bioterrorism and the emergence of multiply-antibiotic resistant strains of Y. pestis increases the urgency for a more detailed understanding of the host-pathogen relationship at the molecular level that may lead to the design of improved medical countermeasures and diagnostics. We have established mouse and rat models of bubonic plague that incorporate flea-to-rodent transmission to investigate the role of specific Y. pestis virulence factors and to characterize the host response to naturally acquired infection. We have characterized the kinetics, microbiology, and histopathology of bubonic plague in rats following intradermal injection of Y. pestis, and used this model to characterize the gene expression profile of Yersinia pestis in the infected lymph node during bubonic plague using whole-genome microarray technology. Our previous work has shown that three important Y. pestis virulence factors, Ail (a Y. pestis outer surface protein), the Type III secretion system (T3SS) encoded on the Yersinia virulence plasmid, and the plasminogen activator (Pla) encoded on the 9.5-kb Y. pestis-specific plasmid all act to limit the polymorphonuclear leukocyte response to bubonic plague infection in vivo (polymorphonuclear leukocytes, also referred to as PMNs or neutrophils, are phagocytic cells that are an important innate defense against infection). Thus, we now have several lines of evidence that the PMN response correlates with successful outcome to infection, and this aspect of host-pathogen interaction has become a focus of our lab. We discovered that the Y. pestis T3SS effector protein YopJ strongly inhibits the secretion of the IL-8 by human neutrophils (submitted for publication during fiscal year 2015). In addition, we reported that a small but significant percentage of Y. pestis, even T3SS-negative attenuated strains, survive and eventually replicate within phagosomes after being ingested by neutrophils. Infected neutrophils were also demonstrated to be taken up by macrophages in vitro, where Y. pestis could continue to replicate. The results indicate that neutrophil phagocytosis is not invariably fatal to Y. pestis, and that virulence factors other than the T3SS that counteract the strongly bactericidal environment of the neutrophil phagosome are important to plague pathogenesis. This has been the focus of research of a post-baccalaureate fellow during the current year. During the past year, we used intravital imaging methodologies to examine the mammalian host response to Y. pestis in the skin immediately after transmission by its natural vector, the rat flea Xenopsylla cheopis, to observe differences relative to the response to needle-inoculated bacteria. Our results showed that uninfected flea bites induce minimal inflammation, but flea-transmitted Y. pestis cause the recruitment of neutrophils roughly in proportion to the number of bacteria deposited in the skin. We observed interactions of flea-transmitted bacteria with macrophages, a cell type much more permissive than neutrophils for survival and growth of Y. pestis. We found that dendritic cells, important sentinel antigen presenting cells, were recruited to, but had minimal interaction with, flea-transmitted bacteria. Additionally, we found that Y. pestis could disseminate from the flea bite site to the draining lymph node and spleen as early as 1 h after flea feeding, significantly earlier than has been previously reported. Our lab previously reported that the draining lymph nodes of rats infected with virulence plasmid-negative, but not wild-type, Y. pestis strains contain elevated levels of IL-17, a proinflammatory cytokine that enhances the neutrophilic response that correlates with control of Y. pestis infection. This year we investigated the interaction of Y. pestis with recently described motile, IL-17-producing &#947;&#948; T cells (&#947;&#948; T17) present in large numbers in the dermis and peripheral lymph nodes that have been shown to be important in the innate immune response to dermal infection. These studies were done in collaboration with Dr. Jason Cyster, a Howard Hughes Investigator at UCSF and the co-discoverer of this cell type. Our experience with intravital microscopy has guided the implementation of new experimental strategies to identify and characterize mechanisms of Y. pestis dissemination from the dermis and resistance to the innate immune response. For example, we are now examining the interaction of Y. pestis with lymph node subcapsular sinus macrophages, the first innate immune cell encountered by the bacteria as they disseminate from the dermis and enter the draining lymph node.